U.S. patent application number 11/328855 was filed with the patent office on 2006-11-02 for chimeric il-10.
This patent application is currently assigned to Beth Israel Deaconess Medical Center, Inc., a Massachusetts corporation. Invention is credited to Alan W. Steele, Terry B. Strom, Xin Xiao Zheng.
Application Number | 20060246032 11/328855 |
Document ID | / |
Family ID | 23397668 |
Filed Date | 2006-11-02 |
United States Patent
Application |
20060246032 |
Kind Code |
A1 |
Strom; Terry B. ; et
al. |
November 2, 2006 |
Chimeric IL-10
Abstract
Disclosed are chimeric proteins having a cytokine fused to an
enzymatically inactive polypeptide which increases the circulating
half-life of the cytokine. The chimeric proteins are useful for
treating, inhibiting, or preventing a variety of conditions,
including septic shock, granulomatous disorders, Type I diabetes,
and various cancers (e.g., multiple myeloma) in a patient.
Inventors: |
Strom; Terry B.; (Brookline,
MA) ; Zheng; Xin Xiao; (Wellesley, MA) ;
Steele; Alan W.; (Wellesley, MA) |
Correspondence
Address: |
FISH & RICHARDSON PC
P.O. BOX 1022
MINNEAPOLIS
MN
55440-1022
US
|
Assignee: |
Beth Israel Deaconess Medical
Center, Inc., a Massachusetts corporation
|
Family ID: |
23397668 |
Appl. No.: |
11/328855 |
Filed: |
January 10, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10145517 |
May 14, 2002 |
7018626 |
|
|
11328855 |
Jan 10, 2006 |
|
|
|
08968905 |
Nov 6, 1997 |
6403077 |
|
|
10145517 |
May 14, 2002 |
|
|
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08355502 |
Dec 12, 1994 |
6410008 |
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08968905 |
Nov 6, 1997 |
|
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Current U.S.
Class: |
424/85.2 ;
424/146.1; 424/94.5 |
Current CPC
Class: |
Y02A 50/423 20180101;
C07K 14/52 20130101; C07K 2317/71 20130101; A61K 38/45 20130101;
A61K 38/38 20130101; C07K 2319/30 20130101; C07K 16/46 20130101;
A61K 38/2066 20130101; Y10S 514/866 20130101; C12N 15/62 20130101;
C07K 2319/00 20130101; C07K 2319/02 20130101; Y02A 50/30 20180101;
A61K 38/2026 20130101; C07K 2319/75 20130101; A61K 38/2026
20130101; A61K 2300/00 20130101; A61K 38/2066 20130101; A61K
2300/00 20130101; A61K 38/38 20130101; A61K 2300/00 20130101; A61K
38/45 20130101; A61K 2300/00 20130101 |
Class at
Publication: |
424/085.2 ;
424/146.1; 424/094.5 |
International
Class: |
A61K 38/20 20060101
A61K038/20; A61K 38/48 20060101 A61K038/48; A61K 39/395 20060101
A61K039/395 |
Claims
1. A method for treating, or inhibiting the onset of, an
immunological disorder in a patient, the method comprising
administering to the patient a therapeutically effective amount of
a chimeric protein comprising interleukin-4 (IL-4) or
interleukin-10 (IL-10) and a polypeptide that increases the
circulating half-life of the IL-4- or IL-10-containing chimera
relative to that of IL-4 or IL-10 alone.
2. The method of claim 1, wherein the polypeptide comprises a hinge
region of an IgG molecule.
3. The method of claim 1, wherein the polypeptide comprises
albumin, or a porcine or rodent glycosyltransferase or
.alpha.-1,3-galactosyltransferase.
4. The method of claim 1, wherein the polypeptide comprises the Fc
region of an IgG molecule but lacks an IgG variable region.
5. The method of claim 4, wherein the polypeptide further comprises
a hinge region of an IgG molecule.
6. The method of claim 4, wherein the Fc region is lytic.
7. The method of claim 4, wherein the Fc region is non-lytic.
8. The method of claim 4, wherein the Fc region includes a mutation
that inhibits complement fixation and high affinity binding to an
Fc receptor by the protein.
9. The method of claim 1, wherein the chimeric protein is
administered to the patient with a pharmaceutically acceptable
carrier.
10. The method of claim 1, wherein the immunological disorder
comprises granuloma formation.
11. The method of claim 1, wherein the immunological disorder is
schistosomiasis.
12-21. (canceled)
22. A chimeric protein comprising interleukin-4 (IL-4) or
interleukin-10 (IL-10) and a polypeptide that increases the
circulating half-life of the IL-4- or IL-10-containing chimera
relative to that of IL-4 or IL-10 alone.
23. The chimeric protein of claim 22, wherein the polypeptide
comprises a hinge region of an IgG molecule.
24. The chimeric protein of claim 22, wherein the polypeptide
comprises albumin, or a porcine or rodent glycosyltransferase or
.alpha.-1,3-galactosyltransferase.
25. The chimeric protein of claim 22, wherein the polypeptide
comprises the Fc region of an IgG molecule but lacks an IgG
variable region.
26. The chimeric protein of claim 25, wherein the polypeptide
further comprises a hinge region of an IgG molecule.
27. The chimeric protein of claim 25, wherein the Fc region is
lytic.
28. The chimeric protein of claim 25, wherein the Fc region is
non-lytic.
29. The chimeric protein of claim 25, wherein the Fc region
includes a mutation that inhibits complement fixation and high
affinity binding to the Fc receptor by the protein.
30. A composition comprising the chimeric protein of claim 22 and a
pharmaceutically acceptable carrier.
Description
CROSS REFERENCE TO RELATED APPLICATION
[0001] This patent application is a continuation of U.S. patent
application Ser. No. 10/145,517, filed on May 14, 2002, which is a
continuation of U.S. patent application Ser. No. 08/968,905, filed
on Nov. 6, 1997, now U.S. Pat. No. 6,403,077, which is a
continuation in part of U.S. patent application Ser. No.
08/355,502, filed on Dec. 12, 1994, now U.S. Pat. No. 6,410,008.
The contents of these prior applications are hereby incorporated by
reference in their entirety for all purposes.
BACKGROUND OF THE INVENTION
[0002] This invention relates to chimeric proteins which include a
cytokine and an enzymatically inactive polypeptide, and therapeutic
uses thereof.
[0003] Cytokines have a wide range of effects on cell growth and
differentiation. The value of certain cytokines has been
recognized, including, for example, IL-2 for promoting the growth
of activated T cells, B cells, LAK cells, and NK cells; IL-3 for
promoting the growth of pluripotent hematopoietic progenitor cells;
granulocyte macrophage-colony stimulating factor (GM-CSF) for
promoting the growth and differentiation of neutrophils and
macrophages, and for activating macrophages; kit ligand for
promoting basophil and mast cell differentiation; IL-4 for
promoting B cell proliferation, enhancing class II gene expression,
enhancing IgG1 and IgE production, and promoting activated T cell
proliferation and effector cell function; IL-5 for enhancing IgA
production and stimulating eosinophil growth; IL-6 for transiently
blocking myeloma growth, inducing immunoglobulin production, and
inducing plasma cell and hepatocyte growth; IL-7 for inducing
immature and mature B and T cell growth; and interferon-a and -a
for their antiviral activity against papilloma viruses, hepatitis
viruses, and herpes virus, and for treating hairy cell leukemia,
myeloma, and other hematopoietic malignancies. Some additional
functions of cytokines are summarized below.
[0004] Reported IL-1 activities include activation of T cells;
induction of IL-2 receptor expression and cytokine gene expression;
enhancement of collagenase, stromelysin, prostaglandin, and PDGF-AA
synthesis by fibroblasts; co-stimulation of thymocyte
proliferation; stimulation of pre-B cell differentiation;
co-stimulation of B cell proliferation and Ig secretion;
augmentation of IL-2 and IFN-induced activation of NK-mediated
cytotoxicity; induction of adhesion molecule expression by
endothelial cells; osteoblast and endothelial cell activation;
enhancement of collagen production by epidermal cells; modulation
of reparative functions following tissue injury; induction of
insulin secretion; and .beta.-islet cell cytotoxicity.
[0005] IL-1 has also been shown to stimulate the release of factors
associated with the growth and differentiation of cells from
myeloid and lymphoid lineages in vitro. IL-1 is thought to induce
the production of granulocyte colony stimulating factor (G-CSF) and
macrophage colony stimulating factor (M-CSF) by human marrow
stromal cells; induce the production of GM-CSF and G-CSF by human
dermal fibroblasts; and induce the production of GM-CSF by human
peripheral blood lymphocytes. IL-1 also stimulates hematopoiesis by
up-regulating receptors for colony stimulating factors and inducing
the proliferation of pluripotent progenitors in the bone marrow.
IL-1 has been shown to protect mice from otherwise lethal doses of
radiation which indicates that this protein is useful in cancer
therapy. Also, IL-1 has been shown to accelerate wound healing,
presumably due to its ability to induce angiogenesis and fibroblast
activation.
[0006] IL-2 has been reported to participate in the activation,
tumoricidal activity, and growth of T cells, NK cells, and LAK
cells; augment B cell growth and immunoglobulin production; augment
IFN-.gamma. production; induce IL-6 production by human monocytes;
modulate histamine release by stimulated basophils; and modulate
expression of the IL-2 receptor. Applications of IL-2 include
anti-tumor therapy employing IL-2-activated LAK and TIL cell
infusions; augmentation of IL-2 levels in treating immunodeficiency
disorders, and increasing of NK cell activity following bone marrow
transplant.
[0007] Reported functions of IL-3 include stimulating the
proliferation of mast cell lines; stimulating the formation of
neutrophils, macrophages, megakaryocytes, basophils, eosinophils,
and mast cells from isolated hematopoietic progenitors; enhancing
growth of certain human T lymphocytes; and potentiating the
activity of eosinophils, basophils, and monocytes. It has been
shown that IL-3 exerts its ability to support multi-lineage colony
formation early in the development of multipotent progenitors. IL-3
exhibits synergy with Stem Cell Factor (kit ligand) in inducing
human CD34.sup.+ cells to form basophils and mast cells. IL-3 has
been used successfully in combination with factors such as GM-CSF
to stimulate hematopoiesis in primates. In addition, sequential
administration of IL-3 and IL-6 in primates stimulates
thrombopoiesis. In vitro studies suggest that IL-3 can be used to
reverse the hematopoietic toxicity associated with AZT treatment.
Recombinant IL-3 has also been used in clinical trials in
combination with other colony stimulating factors as a treatment
for aplastic anemia.
[0008] IL-4 has been reported to be useful for up-regulating MHC
Class II expression in resting B cells; enhancing IgG1, IgE, and
sIgM production by B cells; up-regulating Fc receptor expression
for IgE on B cells and monocytes; increasing viability and growth
of normal resting T cells and certain T cell lines; co-stimulating
growth in certain mast cell lines; maintaining
Lyt-2.sup.-/L3T4.sup.- thymic stem cells; promoting thymocyte
maturation; enhancing the proliferation of granulocyte-macrophage
progenitors, erythrocyte progenitors, and megakaryocytes in
response to G-CSF, EPO, and IL-1, respectively; inhibiting human
breast carcinoma cell growth in culture; inducing progression in B
cells; inducing tumoricidal activity in cultured macrophages; and
regulating adhesion molecule expression on endothelial cells.
[0009] Additionally, IL-4 is thought to act in combination with
IL-1 as an autocrine growth factor for antigen-specific T cells to
enhance antigen presentation and phagocytosis in macrophages. IL-4
not only enhances the development of cytotoxic T lymphocytes (CTL)
from resting murine cells, but it also induces LAK activity. As a
multifunctional cytokine that is reported to augment certain T and
B cell responses, the therapeutic functions of IL-4 include
reconstitution of cellular and humoral immune function following
bone marrow transplantation; induction of terminal differentiation
of acute lymphoblastoid leukemias; amelioration of immunodeficiency
associated with hyper IgM; inhibition of the growth of solid tumors
and B cell lymphomas; and reduction of inflammatory processes
through down-regulation of production of IL-1, TNF, and IL-6. IL-4
has also been used in preclinical models to treat T cell-dependent
autoimmune diseases, e.g., autoimmune diabetes mellitus and
experimental and T cell-dependent allergic encephalomyelitis
(Rapoport et al., 1993, J. Exp. Med. 178:87-99 and Racke et al.,
1994, J. Exp. Med. 180:1961). Hence, IL-4 may be used in treating a
variety of pathologies involving T cell-dependent immune
activities.
[0010] IL-5 has been shown to induce eosinophil colonies in human
liquid bone marrow cultures to induce antibody-mediated killing of
tumor cells by peripheral blood eosinophils. IL-5 also has been
shown to stimulate murine B cells to differentiate and proliferate
and to simulate IgA and IgM secretion in B cells. IL-5 is useful
for treating pathologies related to alterations in eosinophil
activity. For example, suggested uses of IL-5 include treatment of
schistosomiasis (see, e.g., Sanderson, April, 1989, "International
Conference on the Clinical Impact of Interleukins" at the Royal
College of Physicians in London). Other reports suggest the use of
IL-5 in treating patients having certain tumors (see, e.g., Kolb et
al., 1979, Br. J. Cancer 40:410; Pretlow et al., 1983, Cancer Res.
43:2997; and Iwasaki et al., 1986, Cancer 58:1321).
[0011] IL-6 is reported to exhibit multiple functions, including
induction of proliferation in a number of cells, including
EBV-transformed B cells, T cells, mesangial cells, and
keratinocytes; enhancement of the IL-3-dependent proliferation of
multipotential hematopoietic progenitors; promotion of
megakaryocyte maturation; triggering of neuronal differentiation;
growth inhibition of certain melanoma cell lines, myeloid leukemic
cell lines, and breast carcinoma cell lines; induction of B cell
differentiation; stimulation of IgG secretion; and induction of
cytotoxic T cell differentiation.
[0012] Additionally, IL-6 acts on murine thymocytes to induce the
differentiation of Lyt-2.sup.+ CTL in the presence of IL-2, and
IL-6 supports the proliferation of Con-A or T cell receptor
antibody-stimulated T cells in vitro. IL-6 has also been reported
to co-stimulate thymocyte proliferation and induce the release of
acute phase reactants from hepatocytes. IL-6 is also thought to be
an autocrine growth factor for tumor cells from patients with
multiple myeloma.
[0013] IL-7 has been reported to have T cell growth factor
activity. Stem cell factor synergizes with IL-7 to stimulate
proliferation of early T cell progenitors. Also, IL-7 acts as a
co-stimulus with Con A to induce the proliferation of purified
murine T cells. IL-7 has also been reported to induce proliferation
of human peripheral blood T lymphocytes in the presence of
sub-mitogenic doses of Con A and PHA. Certain studies suggest that
IL-7 acts directly on human CD8.sup.+ T cells to augment
cytotoxicity and that IL-7 is a potent differentiation factor for
the development of CTL.
[0014] In mice, IL-7 has been shown to act on CD8.sup.+ T cells to
induce CTL in an IL-2- and IL-6-dependent manner. IL-7 is required
for the IL-1-induced proliferation of murine thymocytes. IL-7 has
further been shown to induce LAK activity from CD8.sup.+ cells
prepared from murine peripheral lymphoid tissues. IL-7 has been
shown to increase surface expression of the ICAM-1 molecule on
melanocytes and melanoma cells. Injection of IL-7 into mice leads
to a 3- to 5-fold increase in circulating immature B cells with a
concurrent 90% reduction in myeloid progenitors in the bone marrow
and a 15-fold increase in myeloid progenitors in the spleen. Hence,
IL-7 is thought to have a similar spectrum of therapeutic
activities in vivo as those reported for IL-2.
[0015] IL-9 stimulates the proliferation of mouse erythroid
progenitors and promotes erythroid differentiation of cells in the
presence of erythropoietin and IL-3 (Bourett et al., 1992, Exp.
Hematol. 20:868). IL-9 has also been shown to enhance the survival
of T cell lines in vitro (Renaud et al., 1990, Cytokine 2:9). IL-9
also potentiates IL-4-dependent Ig production by human B
lymphocytes, and it promotes IL-6 production by murine mast cells
lines derived from bone marrow. In addition, IL-9 is involved in
the differentiation of hippocampal progenitors (Uyttenhove et al.,
1991, J. Exp. Med. 173:519).
[0016] IL-10 is a cytokine produced by activated Th2 cells, B
cells, keratinocytes, monocytes, and macrophages (Moore et al.,
1993, Annu. Rev. Immunol. 11:165). IL-10 can be used to stimulate
growth and differentiation of activated human B cells. In vitro,
murine and human IL-10 inhibit cytokine synthesis (e.g.,
IFN-.gamma. TNF-.beta., and IL-2) by Th1 cells, NK cells,
monocytes, and macrophages (Fiorentino et al., 1989, J. Exp. Med.,
170:2081-2095; Fiorentino et al., 1991, J. Immunol. 146:3444; Hsu
et al., 1992, Int. Immunol. 4:563; Hsu et al., 1992, Int. Immunol.
4:563; D'Andrea et al., 1993, J. Exp. Med. 178:1041; and de Waal
Malefyt et al., 1991, J. Exp. Med. 174:915; Fiorentino et al.,
1991, J. Immunol. 147:3815). Thus IL-10 is useful for inhibiting
Th1 responses to prevent transplant rejection and T cell-mediated
autoimmune diseases, such as type I diabetes and multiple
sclerosis. The ability of IL-10 to inhibit secretion of the
pro-inflammatory cytokines (e.g., IL-1, IL-6, IL-8, and
TNF-.alpha.) suggests that IL-10 is a useful anti-inflammatory
agent in the treatment of rheumatoid arthritis and psoriasis.
[0017] IL-10 has been recognized for its value in treating
septicemia. Gram-negative septicemia in hospitalized patients is
invariably associated with high morbidity and mortality (Bone,
1991, Ann. Intern. Med. 115:457). Case fatality rates of 20-60%
reflect the frequent development of acute lung injury (Byrne et
al., 1987, Acute Care 13:206) and multiple organ failure (Abrams et
al., 1989, Surg. Rounds 12:44), as well as the lack of effective
therapies. Endotoxin (LPS), a product of gram-negative bacteria, is
a major causative agent in the pathogenesis of septic shock
(Glausner et al., 1991, Lancet 338:732). A septic shock-like
syndrome can be induced experimentally by a single injection of LPS
into animals. Injection of IL-10 into mice inhibits secretion of
tumor necrosis factor in vivo and protects against the lethal
effects of endotoxin (Gerard et al., 1993, J. Exp. Med. 177(2):547;
de Waal Malefyt et al., 1991, J. Exp. Med. 174:915; Fiorentino et
al., 1991, J. Immunol. 147:3815; and Moore et al., 1990, Science
248:1230).
[0018] Upon infection with Schistosoma, a genus of flatworms, the
organism deposits its eggs into the liver, causing granuloma
formation and fibrosis of liver tissue. Liver damage caused by
Schistosome infection can lead to cirrhosis of the liver.
Schistosomiasis is often chronic and debilitating.
[0019] Naturally-occurring cytokines have short circulating
half-lives; for example, naturally-occurring IL-10 is
therapeutically effective for approximately 30 minutes following
administration (Gerard et al., 1993, J. Exp. Med. 177(2):547).
SUMMARY OF THE INVENTION
[0020] I have discovered that the in vivo half-life of a cytokine
can be increased by bonding the cytokine to a polypeptide which
increases the longevity of the cytokine while being enzymatically
inactive in humans, and I have discovered that certain of the
chimeric cytokines (i.e., chimeric proteins or chimeras) are useful
for treating or inhibiting the onset of conditions such as septic
shock, granulomatous disorders (e.g., schistosomiasis), Type I
diabetes, certain cancers (e.g., multiple myeloma), and chronic
infections.
[0021] Accordingly, in one aspect, the invention features a
chimeric protein having a cytokine bonded to a polypeptide which is
enzymatically inactive in humans and which increases the
circulating half-life of the cytokine in vivo by a factor of at
least 2, and preferably by a factor of at least 10.
[0022] Useful enzymatically inactive polypeptides include not only
proteins that are not enzymes, such as albumin, but also enzymes
that have enzymatic activity in an organism other than humans but
which are inactive in humans. For example, useful polypeptides
include plant enzymes, porcine or rodent glycosyltransferases, and
.alpha.-1,3-galactosyltransferases (see, e.g., Sandrin et al.,
1993, PNAS 90:11391).
[0023] The enzymatically inactive polypeptide can include an IgG
hinge region and a half-life increasing polypeptide. In this
embodiment, the IgG hinge region is bonded to the cytokine and
serves as a flexible polypeptide spacer between the cytokine and
the half-life-increasing polypeptide (e.g., IgG Fc or albumin).
[0024] When the enzymatically inactive polypeptide includes an IgG
hinge region and the Fc region of an IgG molecule, it lacks an IgG
variable region of a heavy chain so that the binding specificity
conferred by the variable region is lacking in the chimera. The Fc
region can include a mutation which inhibits complement fixation
and prevents Fc from binding the Fc receptor with high affinity,
thus preventing the chimera from being lytic. Alternatively, the Fc
region can be lytic, i.e., be able to bind complement and bring
about lysis of the cell to which the chimera binds.
[0025] The cytokine portion of the chimeric protein can be an
interleukin, such as IL-10, IL-6, IL-4, IL-1, IL-2, IL-3, IL-5,
IL-7, IL-8, IL-9, IL-12, or IL-15. Other useful cytokines include
GM-CSF, G-CSF, interferons (e.g., IFN-.alpha., IFN-.beta., and
IFN-.gamma.), and tumor necrosis factors (e.g., TNF-.alpha. and
TNF-.beta.).
[0026] A chimeric cytokine of the invention can be used in a
therapeutic composition formed by admixture of the chimeric protein
with a pharmaceutically acceptable carrier. In various embodiments,
the invention provides methods for treating or inhibiting the
development of a variety of conditions. For example, the IL-10
chimeras and IL-4 chimeras can each be used to treat or inhibit
granuloma formation (e.g., schistosomiasis). IL-10 chimeras and
IL-4 chimeras are also useful for inhibiting the development of
Type I diabetes, for treating or inhibiting the development of
Crohn's disease, ulcerative colitis, or Boeck's disease. The IL-10
chimera is useful for treating or inhibiting septic shock. IL-6
chimeras are useful for treating or inhibiting the development of
multiple myeloma in a patient. TNF-.alpha. chimeras and TNF-.beta.
chimeras each are useful for combatting cervical cancer caused by
papilloma viruses, liver cancer caused by hepatitis viruses, and
skin eruptions caused by herpes viruses.
[0027] By "cytokine" is meant any of the non-antibody proteins
released by one cell population (e.g., primed T-lymphocytes) on
contact with specific antigen, which act as intercellular
mediators, as in the generation of an immune response. One
important class of cytokines are those which induce proliferation
of lymphocytes, e.g., T cells.
[0028] By IgG "Fc" region is meant a naturally-occurring or
synthetic polypeptide homologous to the IgG C-terminal domain that
is produced upon papain digestion of IgG. IgG Fc has a molecular
weight of approximately 50 kD. In the molecules of the invention,
the entire Fc region can be used, or only a half-life enhancing
portion. In addition, many modifications in amino acid sequence are
acceptable, as native activity is not in all cases necessary or
desired.
[0029] By "non-lytic" IgG Fc is meant an IgG Fc region which lacks
a high affinity Fc receptor binding site and which lacks a C'1q
binding site. The high affinity Fc receptor binding site includes
the Leu residue at position 235 of IgG Fc; the Fc receptor binding
site can be functionally destroyed by mutating or deleting Leu 235.
For example, substitution of Glu for Leu 235 inhibits the ability
of the Fc region to bind the high affinity Fc receptor. The C'1q
binding site can be functionally destroyed by mutating or deleting
the Glu 318, Lys 320, and Lys 322 residues of IgG1. For example,
substitution of Ala residues for Glu 318, Lys 320, and Lys 322
renders IgG1 Fc unable to direct ADCC.
[0030] By "lytic" IgG Fc is meant an IgG Fe region which has a high
affinity Fe receptor binding site and a C'1q binding site. The high
affinity Fc receptor binding site includes the Leu residue at
position 235 of the IgG Fc. The C'1q binding site includes the Glu
318, Lys 320, and Lys 322 residues of IgG1. Lytic IgG Fc has
wild-type residues or conservative amino acid substitutions at
these binding sites. Lytic IgG Fc can target cells for antibody
dependent cellular cytotoxicity (ADCC) or complement directed
cytolysis (CDC).
[0031] By IgG "hinge" region is meant a polypeptide homologous to
the portion of a naturally-occurring IgG which includes the
cysteine residues at which the disulfide bonds linking the two
heavy chains of the immunoglobulin form. For IgG1, the hinge region
also includes the cysteine residues at which the disulfide bonds
linking the .gamma.1 and light chains form. The hinge region is
approximately 13-18 amino acids in length in IgG1, IgG2, and IGg4;
in IgG3, the hinge region is approximately 65 amino acids in
length.
[0032] By polypeptide "spacer" is meant a polypeptide which, when
placed between the half-life-increasing polypeptide and a cytokine,
possesses an amino acid residue with a normalized B value
(B.sub.norm; a measure of flexibility) of 1.000 or greater,
preferably of 1.125 or greater, and, most preferably of 1.135 or
greater (see, e.g., Karplus et al., 1985, Naturwissenschaften
72:212). Amino acids which are commonly known to be flexible
include glutamic acid, glutamine, threonine, lysine, serine,
glycine, proline, aspartic acid, asparagine, and arginine.
[0033] The invention offers several features and advantages: (1)
the chimeric proteins of the invention have an extended circulating
half-life and provide long term protection; (2) because many of the
cytokines and longevity-increasing polypeptides useful in the
invention have already been purified, the chimeric proteins can
easily be purified by employing methods that have been described
for purifying the cytokine or longevity-increasing polypeptide; (3)
some of the chimeric proteins are mutated such that they are
defective for antibody-dependent cell-mediated cytotoxicity (ADCC)
and complement directed cytolysis (CDC), thus making them useful
for treating or inhibiting the onset of septic shock, type I
diabetes, or multiple myeloma without destroying the target
cells.
[0034] An additional advantage of chimeric proteins that include an
Fc polypeptide is that they cannot cross the blood/brain barrier
and enter the brain where polypeptides such as IL-6, tumor necrosis
factor, IL-1.alpha., and IL-1.beta., could cause side effects by
reacting with regulatory centers in the brain. Among the side
effects caused by these cytokines in the absence of an Fc
polypeptide are somnolence, fever, and hypotension.
[0035] Other features and advantages of the invention will be
apparent from the following description of the preferred
embodiments thereof, and from the claims.
DETAILED DESCRIPTION
[0036] The drawings will first be briefly described.
DRAWINGS
[0037] FIG. 1 is a schematic representation of the scheme used for
the genetic fusion of murine IL-10 and murine Fc.gamma.2a cDNAs to
create a murine IL-10/Fc chimeric cytokine. Mutations were made in
the CH2 domain of a Fc.gamma.2a fragment with site-directed
mutagenesis to replace Glu 318, Lys 320, and Lys 322 with Ala
residues; Leu 235 was replaced with Glu to render the IL-10/Fc
chimeric protein ineffective in directing ADCC and CDC. The
non-lytic chimeric protein is referred to hereinafter as
"IL-10/Fc." The lytic chimeric protein (without the mutation) is
referred to as "IL-10/Fc++."
[0038] FIGS. 2A-B are reproductions of blots obtained by Western
blot analysis of the IL-10/Fc chimeric protein. The
SDS-polyacrylamide gels were run under reducing (lanes 2-4) and
non-reducing (lanes 5-7) conditions. Western blotting was performed
with antibodies directed against mIgG Fc (FIG. 2A), or mIL-10 (FIG.
2B). For both FIG. 2A and FIG. 2B, lane 1 was loaded with a high
molecular weight protein standard; lanes 2 and 5 were loaded with
mIgG2a; lanes 3 and 6 were loaded with IL-10/Fc++; and lanes 4 and
7 were loaded with IL-10/Fc.
[0039] FIG. 3 is a histogram demonstrating that rIL-10 (wild-type,
recombinant IL-10) and IL-10/Fc inhibit LPS-induced production of
IL-6 by macrophages. PU5-1.8 cells (10.sup.6 cells/ml) were
pre-incubated with various concentrations of IL-10/Fc or rIL-10, as
indicated, for 24 hours. LPS (10 ig/ml) then was added, and the
cells were incubated for an additional 24 hours. Supernatants were
collected and the IL-6 concentration was assayed by ELISA.
[0040] FIG. 4 is a histogram showing the co-stimulatory effects of
IL-10/Fc on mast cell proliferation. The ability of rIL-10 or
IL-10/Fc to enhance IL-4-dependent growth of MC/9 mast cells was
assessed in a [.sup.3H] thymidine incorporation assay. MC/9 mast
cells (5.times.10.sup.3 cells/ml) were cultured for 3 days with
rIL-10 (100 U/ml), IL-10/Fc (100 U/ml), rIL-4 (100 U/ml), or
combinations of these factors in the presence or absence of a
neutralizing anti-murine IL-10 mAb as indicated.
[0041] FIG. 5 is a plot of the IL-10/Fc circulating half-life. The
time-related serum concentration of IL-10/Fc was determined
following a single bolus intravenous dose (8 .mu.g) of the chimeric
protein. Blood samples were obtained by retro-orbital bleeding at
the indicated intervals. IL-10/Fc levels were detected by ELISA
with a rat-anti-mouse IL-10 mAb as the capture antibody and
horseradish peroxide-conjugated rat anti-mouse IgG heavy chain mAb
as the detection antibody.
[0042] FIGS. 6A-6D are FACS profiles indicating that, as is
desired, IL-10/Fc exhibits poor Fc.gamma.R I binding activity.
Fc.gamma.R I binding assays were performed using human Fc.gamma.R I
cDNA transfected CHO cells (murine Fc.gamma.R I, Fc.gamma.R II, and
IL-10 receptor negative). The Fc.gamma.R I binding ability of PBS
(FIG. 6A), mIgG2a (FIG. 6B), IL-10/Fc++ (FIG. 6D), and IL-10/Fc
(FIG. 6C) was analyzed by FACS.
[0043] FIG. 7 is a plot showing that IL-10/Fc confers prolonged
protection from the lethal effects of LPS following an injection of
500 .mu.g LPS. This plot shows the survival rates for the following
six groups of BALB/c mice: (i) 12 mice which were treated for 30
minutes with rIL-10; (ii) 12 mice which were treated for 30 minutes
with 2,000 U of IL-10/Fc; (iii) 12 mice which were treated for 24
hours with 4,000 U of IL-10/Fc; (iv) 12 mice which were treated for
30 minutes with phosphate buffered saline; (v) 6 mice which were
treated for 30 minutes with 0.6 .mu.g mIgG2a; and (vi) 12 mice
which were treated for 24 hours with 4,000 U rIL-10.
[0044] FIG. 8 is a histogram which indicates that IL-10/Fc
down-regulates granuloma size in livers of Schistosome-infected
mice.
[0045] FIG. 9 is a graph which shows that IL-10/Fc prevents the
onset of diabetes in non-obese diabetic (NOD) mice.
[0046] FIG. 10 is a graph which indicates that IL-10/Fc prevents
the onset of diabetes in NOD mice.
[0047] FIG. 11 is a graph which indicates that mice treated with
IL-10/Fc bear suppressor (anti-diabetogenic) lymphocytes.
[0048] FIG. 12 is a schematic representation of the scheme used for
the genetic fusion of murine IL-4 and murine Fc.gamma.2a cDNAs to
create a non-lytic murine IL-4/Fc chimeric cytokine. Mutations were
made in the CH2 domain of a Fc.gamma.2a fragment with site-directed
mutagenesis to replace Glu 318, Lys 320, and Lys 322 with Ala
residues; Leu 235 was replaced with Glu to render the IL-4/Fc
chimeric protein ineffective in directing ADCC and CDC. The
non-lytic chimeric protein is referred to hereinafter as "IL-4/Fc."
The lytic chimeric protein (without the mutation) is referred to as
"IL-4/Fc++."
[0049] FIG. 13 is a plot of the .sup.3H incorporated into cells in
a CTLL-2 proliferation assay versus the concentration of
recombinant IL-4 or IL-4/Fc in the assay. Recombinant IL-4 and
IL-4/Fc were used at equivalent molar concentrations of IL-4, as
determined by ELISA.
[0050] FIG. 14 is a graph which indicates that the circulating
half-life of non-lytic IL-4/Fc is 25 hours.
[0051] FIG. 15 is a histogram showing that non-lytic IL-4/Fc
down-regulates granuloma size in livers of Schistosome-infected
mice.
ABBREVIATIONS
[0052] The following abbreviations are used herein: TABLE-US-00001
ADCC antibody dependent cell-mediated cytotoxicity CDC complement
directed cytolysis CMV cytomegalovirus Con A concanavalin A GM-CSF
granulocyte macrophage-colony stimulating factor HBSS Hank's
balanced salt solution IL interleukin NOD non-obese diabetic PBS
phosphate-buffered saline TNF tumor necrosis factor
[0053] Before providing detailed working examples of the invention,
I have described some of the parameters of the invention.
[0054] Chimeric Cytokines: Conventional molecular biology
techniques can be used to produce chimeric proteins having a
cytokine (e.g., an interleukin) bonded to an enzymatically inactive
polypeptide (e.g., a lytic or non-lytic Fc region of IgG). Numerous
polypeptides are suitable for use as enzymatically inactive
proteins in the invention. Preferably, the protein has a molecular
weight of at least 10 kD; a net neutral charge at pH 6.8; a
globular tertiary structure; human origin; and no ability to bind
to surface receptors other than a receptor for the cytokine (e.g.,
the IL-10 receptor). Where the enzymatically inactive polypeptide
is IgG, preferably, the IgG portion is glycosylated. If desired,
the enzymatically inactive polypeptide can include an IgG hinge
region positioned such that the chimeric protein has a cytokine
bonded to an IgG hinge region with the hinge region bonded to a
longevity-increasing polypeptide. Thus, the hinge region can serve
as a spacer between the cytokine and the longevity-increasing
polypeptide. A person skilled in molecular biology can readily
produce such molecules from an IgG2a-secreting hybridoma (e.g.,
HB129) or other eukaryotic cells or baculovirus systems. As an
alternative to using an IgG hinge region, a flexible polypeptide
spacer, as defined herein, can be used. Using conventional
molecular biology techniques, such a polypeptide can be inserted
between the cytokine and the longevity-increasing polypeptide.
[0055] Where the enzymatically inactive protein includes an Fc
region, the Fc region can be mutated, if desired, to inhibit its
ability to fix complement and bind the Fc receptor with high
affinity. For murine IgG Fc, substitution of Ala residues for Glu
318, Lys 320, and Lys 322 renders the protein unable to direct
ADCC. Substitution of Glu for Leu 235 inhibits the ability of the
protein to bind the Fc receptor with high affinity. Appropriate
mutations for human IgG also are known (see, e.g., Morrison et al.,
1994, The Immunologist 2: 119-124 and Brekke et al., 1994, The
Immunologist 2: 125). Other mutations can also be used to inhibit
these activities of the protein, and art-recognized methods can be
used to assay for the ability of the protein to fix complement or
bind the Fc receptor. Other useful enzymatically inactive
polypeptides include albumin (e.g., human serum albumin),
transferrin, enzymes such as t-PA which have been inactivated by
mutations, and other proteins with a long circulating half-life and
without enzymatic activity in humans.
[0056] Numerous cytokines have been cloned and are useful in the
invention. Conventional methods can readily be used to subclone a
gene encoding a desired cytokine into a vector for production a
chimeric protein. For example, the murine IL-10 gene has been
described (Moore et al., 1990, Science 248: 1230-1234), and the
human IL-10 gene has been cloned (see, e.g., U.S. Pat. No.
5,231,012, incorporated herein by reference). Human IL-4 has also
been cloned (Yokata et al., 1986, PNAS 83:5894). If desired, the
cytokine can be truncated or mutated as long as it retains a useful
biological function, as determined with conventional methods for
assaying cytokine function.
[0057] Preferably, the enzymatically inactive polypeptide used in
the production of the chimeric protein (e.g., IgG Fc) has, by
itself, an in vivo circulating half-life greater than that of the
cytokine (e.g., IL-10). More preferably, the half-life of the
chimeric protein is at least 2 times that of the cytokine alone.
Most preferably, the half-life of the chimeric protein is at least
10 times that of the cytokine alone. The circulating half-life of
the chimeric protein can be measured in an ELISA of a sample of
serum obtained from a patient treated with the chimeric protein. In
such an ELISA, antibodies directed against the cytokine can be used
as the capture antibodies, and antibodies directed against the
enzymatically inactive protein can be used as the detection
antibodies, allowing detection of only the chimeric protein in a
sample. Conventional methods for performing ELISAs can be used, and
a detailed example of such an ELISA is provided herein.
[0058] The chimeric proteins can be synthesized (e.g., in mammalian
cells) using conventional methods for protein expression using
recombinant DNA technology. Because many of the polypeptides used
to create the chimeric proteins have been previously purified, many
of the previously-described methods of protein purification should
be useful, along with other conventional methods, for purifying the
chimeric proteins of the invention. If desired, the chimeric
protein can be affinity-purified according to standard protocols
with antibodies directed against the cytokine. Antibodies directed
against the enzymatically inactive protein are also useful for
purifying the chimeric protein by conventional immunoaffinity
techniques. If desired, the activity of the chimeric protein can be
assayed with methods that are commonly used to test the activity of
the cytokine alone. It is not necessary that the activity of the
chimeric cytokine be identical to the activity of the cytokine
alone. For example, the chimeric cytokine may be more or less
active than is the cytokine alone.
Therapeutic Use of Chimeric Cytokines:
[0059] The chimeric cytokines of the invention can be used to treat
or inhibit (including completely preventing) the onset of a variety
of conditions in a patient, including septicemia, septic shock,
granuloma formation, Type I diabetes, multiple myeloma, bacterial
or fungal infections, virus-associated cancers (e.g., Burkitt's
lymphoma), and certain other cancers. The therapeutic uses of the
chimeric cytokines are correlated with the therapeutic uses of the
cytokines in the absence of the enzymatically inactive
half-life-increasing polypeptide. Thus, the chimeric cytokines of
the invention can substitute for their corresponding cytokines in
numerous previously described applications, such as those which are
summarized herein.
[0060] Where the cytokine is IL-10, a therapeutic composition that
includes an IL-10 chimera can be administered to a patient to treat
or inhibit septicemia or septic shock. Where the cytokine is IL-10
or IL-4, the chimeric cytokine can be used to treat or inhibit
granulomatous disorders (e.g., schistosomiasis), Crohn's disease
(i.e., regional enteritis), multiple sclerosis, psoriasis,
rheumatoid arthritis, systemic lupus erythematosus, iritis,
inflammatory bowel disease, and Boeck's disease (sarcoidosis). IL-5
chimeras can be used to treat granulomatous disorders such as
schistosomiasis, and IL-5 chimeras can be used to induce
antibody-mediated killing of tumor cells by peripheral blood
eosinophils. These chimeras are also useful for enhancing IgA
production and stimulating eosinophil growth. Where the cytokine is
IL-6, the chimeric cytokine is particularly useful for treating
multiple myelomas, breast carcinomas, and melanomas; and inducing
neuronal, cytotoxic T cell, and B cell differentiation. IL-6
chimeras are also useful for their anti-viral activity, and for
inducing hybridoma and hepatocyte growth. Chimeric cytokines that
include a tumor necrosis factor (e.g., TNF-.alpha. or TNF-.beta.)
are useful for treating or inhibiting cervical cancers that are
associated with papilloma virus infections, liver cancers that are
associated with hepatitis virus infections, and skin eruptions that
are associated with herpes virus infections.
[0061] IL-1, IL-2, IL-3, IL-6, and IL-15 can be used as growth
factors for hematological deficiencies, and they are useful
protecting against side effects, or facilitating recovery, in
irradiation therapy or chemotherapy. Other examples of the
functions of the chimeric proteins include IL-2 or IL-15 chimeras
for promoting the growth of activated T cells, B cells, LAK cells,
and NK cells. IL-3 chimeras are useful for promoting the growth of
pluripotent hematopoietic progenitor cells and reversing the
hematopoietic toxicity associated with AZT treatment. IL-3 chimeras
can also be used to treat aplastic anemia. GM-CSF chimeras are
useful for promoting the growth and differentiation of neutrophils
and macrophages, and for activating macrophages. Other chimeric
cytokine activities include IL-7 chimeras for inducing immature B
and T cell growth.
[0062] Where the Fc region of the chimeric protein is lytic, the
chimeric protein is particularly useful for treating multiple
myeloma. Lytic Fc chimeras can be used to deplete the patient of
suppressor lymphocytes and to suppress chronic immunity. Lytic Fc
chimeras can also be used to treat cancers, such as those which are
associated with viruses. For example, lytic Fc chimeras of the
invention can be used to treat renal cell carcinomas, melanomas,
lymphomas, Papilloma virus-associated cervical cancers, or Kaposi's
sarcoma. In addition, the invention can be used to combat liver
cancer caused by infections with hepatitis B or hepatitis C. The
invention is also useful for treating Epstein Barr Virus-associated
lymphomas, such as Burkitt's lymphoma.
[0063] Chimeric cytokines that include a lytic Fc can also be used
to treat chronic infections, such as infections associated with
suppression of the immune system, e.g., Acquired Immune Deficiency
Syndrome (AIDS)-associated infections. Examples of AIDS-associated
infections that are commonly seen include infections with protozoa
(e.g., Pneumocystis carinii, Toxoplasma gondii, and
Cryptosporidium), fungi (e.g., Candida sp. and Cryptococcus
neoformans), viruses (e.g., Cytomegalovirus, Herpes simplex, and
Herpes zoster), or bacteria (e.g., Mycobacterium
avium-intracellulare and Mycobacterium tuberculosis). Such
infections can cause pneumonia, impairment of the central nervous
system, diarrhea, esophagitis, meningitis, retinitis, or
colitis.
[0064] The invention can also be used to treat non-AIDS-associated
infections with various species of mycobacteria. These bacteria are
causative agents in the etiology of a number of conditions. For
example, M. tuberculosis infections cause tuberculosis; M.
abscessus cause a traumatic infection of the knee; M. bovis is the
primary cause of tuberculosis in cattle and is transmissible to
humans and other animals; M. intracellulare is associated with lung
lesions in humans; M. kansasii causes a tuberculosis-like pulmonary
disease and causes infections, and usually lesions, in the spleen,
liver, pancreas, testes, joints, and the lymph nodes; M. leprae
causes leprosy; M. scrofulaceum is associated with inflammation of
the cervix in children, and lesions in leprosy patients; and M.
ulcerans causes Buruli ulcers in humans.
[0065] The invention can also be used to treat leishmaniasis,
including acute and diffuse cutaneous forms, mucocutaneous forms,
and visceral forms. These infections, caused by species such as L.
major, L. tropica, various strains of L. mexicana, L. braziliensis,
and L. donovani, can result in lesions and ulcers over the
body.
[0066] A therapeutic composition that includes a chimeric protein
of the invention can be formulated, according to standard
protocols, by admixture of the chimeric protein and a
pharmaceutically acceptable carrier such as water or saline. If
desired, a combination of chimeric proteins can be administered to
a patient, either sequentially or simultaneously (e.g., treatment
of granuloma formation by administration of IL-10/Fc, followed by
administration of IL-4/albumin). The chimeric protein can be
administered to a patient intravenously, intraperitoneally,
intramuscularly, and/or subcutaneously. Generally, a chimeric
protein dosage of 1 .mu.g/kg body weight to 500 mg/kg body weight
can be used; preferably, the dosage is 10 .mu.g/kg body weight to
100 .mu.g/kg body weight. Preferably, the chimeric protein is
administered before or at the first sign of disease onset; if
desired, the chimeric protein can be administered before signs of
disease appear. Those skilled in the art of medicine will be able
to adjust the dosage and frequency of administration as desired.
Generally, the chimeric proteins will be administered at 12-hour
intervals. The efficacy of the treatment can be determined by
monitoring the patient for commonly-known signs of the disease, or
by assaying fluid (e.g., serum) samples of the patient for the
presence of the chimeric protein.
[0067] Inhibition of Septic Shock: Septic shock in a patient (e.g.,
a human) can be treated or inhibited by administering to the
patient a therapeutically effective amount of a chimeric protein
which has an IL-10 polypeptide bonded to an enzymatically inactive
(in humans) polypeptide which increases the circulating half-life
of the cytokine by a factor of at least 2. For example, a chimeric
protein having an IL-10 polypeptide bonded to an IgG hinge region
with the hinge region bonded to a lytic or non-lytic IgG Fc region
can be used (these polypeptides are referred to herein as
IL-10/Fc++ and IL-10/Fc, respectively). The chimeric protein can be
formulated in a pharmaceutically acceptable carrier, e.g., saline,
for administration (e.g., intravenous administration) to the
patient. Generally, a dosage of 0.01 mg/kg to 500 mg/kg body weight
is sufficient; preferably, the dosage is 10 .mu.g/kg to 100
.mu.g/kg. If desired, the efficacy of the treatment regimen can be
assessed with conventional methods of monitoring patients for
septic shock.
[0068] Treatment is begun with the diagnosis or suspicion of
septicemia or endotoxemia, and treatment is repeated at 12-hour
intervals until stabilization of the patient's condition is
achieved. Such an assessment can be made on the basis of the
observation that serum TNF levels are undetectable by ELISA. The
patient's condition can also be monitored by measuring the level of
the circulating chimeric protein at 4-hour intervals. The chimeric
protein level can be measured using a two-point ELISA in which
antibodies directed against the cytokine are used as the capture
antibodies and antibodies directed against the enzymatically
inactive protein are used as the detection antibodies.
[0069] Inhibition of the Development of Diabetes: The chimeric
IL-10 and IL-4 molecules of the invention can each be administered
to patients (e.g., humans) to treat or inhibit the development of
diabetes. The chimeric protein can be formulated as a therapeutic
composition by admixture of the chimeric protein with a
pharmaceutically acceptable carrier (e.g., saline). Using
conventional methods, such therapeutic compositions can be
formulated for intraperitoneal, intravenous, subcutaneous, or
intramuscular administration. Preferably the therapeutic
composition is administered to the patient upon discovery of
anti-beta cell autoimmunity and/or subtle pre-diabetic changes in
glucose metabolism (i.e., blunted early i.v. glucose tolerance
test), and administration is repeated every other day, or at a
frequency of at least once per week. The preferred dosage of the
chimeric protein can be determined by using standard techniques to
monitor glucose levels, anti-beta cells autoantibody level, or
abnormalities in glucose tolerance tests of the patient being
treated. For humans, a chimeric protein dosage of 1 .mu.g to 500
mg/kg body weight is sufficient. Generally, the preferred dosage is
1 to 200 .mu.g/kg; more preferably, the dosage is approximately 50
.mu.g/kg.
[0070] Treatment of Cancer: The lytic chimeric proteins of the
invention are useful for treating a number of cancers, e.g.,
multiple myeloma, in a human. For example, naturally-occurring
IL-10 is known to inhibit the production of IL-6 and tumor necrosis
factor. Multiple myeloma is a malignant plasma cell disorder in
which IL-6 functions as an autocrine growth factor for many of the
cells involved. In addition, multiple myeloma cells bear IL-10
receptors, and thus the IL-10 portion of an IL-10/FC++ chimeric
protein targets the protein to the cancer cells which are then
lysed by the lytic Fc portion of the chimera. In this aspect of the
invention, a therapeutic composition of a pharmaceutically
acceptable carrier and a chimeric cytokine (e.g., IL-10/Fc++) is
administered to a patient diagnosed with multiple myeloma.
Similarly, other lytic chimeric cytokines can be used to treat
cancers of cells bearing receptors for the cytokine portion of the
chimera.
[0071] There now follow detailed working examples of the use of
chimeric proteins of the invention to prevent (i.e., completely
inhibit the development of) septic shock in a patient, to inhibit
granuloma formation (e.g., schistosomiasis), and to prevent the
onset of diabetes in a patient.
Treatment of Septic Shock with IL-10/Fc
[0072] Genetic Construction of IL-10/Fc: Complementary DNAs for
murine IL-10 and murine Fc.gamma.2a were generated from mRNA
extracted from concanavalin (Con A)-stimulated murine splenic cells
(C57BL/6J; Jackson Laboratory, Bar Harbor, Me.) and an
IgG2a-secreting hybridoma (American Type Culture Collection HB129,
Rockville, Md.), respectively, using standard techniques with
reverse transcriptase MMLV-RT (Gibco BRL, Grand Island, N.Y.) and a
synthetic oligo-dT.sub.(12-18) oligonucleotide (Gibco BRL). The
IL-10 cDNA was then amplified by PCR using IL-10-specific synthetic
oligonucleotides. The 5' oligonucleotide inserted a unique NotI
restriction site 40 nucleotides 5' to the translational start
codon, while the 3' oligonucleotide eliminated the termination
codon and changed the C-terminal serine codon from AGC to TCG to
accommodate the creation of a unique BamHI site at the IL-10/Fc
junction. Synthetic oligonucleotides used for the amplification of
the Fc.gamma.2a domain cDNA changed the first codon of the hinge
from Glu to Asp in order to create a unique BamHI site spanning the
first codon of the hinge and introduce a unique XbaI site 3' to the
termination codon.
[0073] To make the construct for the non-lytic IL-10/Fc
oligonucleotide, site-directed mutagenesis was used to replace Glu
318, Lys 320, and Lys 322 of the C1q binding motif of Fc with Ala
residues. Similarly, Leu 235 was replaced with Glu to inactivate
the Fc.gamma.R I binding site. Ligation of the IL-10 and
Fc.gamma.2a components in the correct translational reading frame
at the unique BamHI site yielded a 1,236 bp long open reading frame
encoding a single 411 amino acid polypeptide (including the 18
amino acid IL-10 signal peptide) with a total of 13 cysteine
residues (FIG. 1). The mature, secreted homodimeric IL-10/Fc is
predicted to have a total of up to eight intramolecular and three
inter-heavy chain disulfide linkages, and a molecular weight of
90.1 kD, not accounting for glycosylation.
[0074] Expression and Purification of IL-10/Fc: Proper genetic
construction of both IL-10/Fc++ (carrying the wild-type Fc.gamma.2a
sequence) and IL-10/Fc was confirmed by DNA sequence analysis
following cloning of the fusion genes as NotI-XbaI cassettes into
the eukaryotic expression plasmid pRc/CMV (Invitrogen, San Diego,
Calif.). This plasmid carries a CMV promoter/enhancer, a bovine
growth hormone polyadenylation signal, and a neomycin resistance
gene for selection against G418. Plasmids carrying the IL-10/Fc++
or IL-10/Fc fusion genes were transfected into Chinese Hamster
Ovary cells (CHO-K1) by electroporation (1.5 kV/3 .mu.F/0.4 cm/PBS)
and selected in serum-free Ultra-CHO media (BioWhittaker Inc.,
Walkerville, Md.) containing 1.5 mg/ml of G418 (Geneticin, Gibco
BRL). After subcloning, clones which produced the chimeric protein
at high levels were selected by screening supernatants by ELISA for
IL-10. IL-10/Fc and IL-10/FC++ chimeric proteins were purified from
culture supernatants by protein-A sepharose affinity chromatography
followed by dialysis against PBS and 0.22 .mu.m filter
sterilization. Purified proteins were stored at -20.degree. C.
until they were used.
[0075] Confirmation of Size, and IL-10 and Fc.gamma.2a Isotype
Specificity: Western blot analysis following SDS-PAGE under
reducing (+DTT) and non-reducing (-DTT) conditions was performed
using monoclonal anti-murine IL-10 (PharMingen) or polyclonal
anti-murine Fc.gamma. primary antibodies (Pierce, Rockford, Ill.).
As is shown in FIG. 2, the IL-10/Fc chimeric proteins each migrated
under reducing (+DTT) conditions as a single species at the
expected molecular size of 45 kD. Under non-reducing (-DTT)
conditions, each IL-10/Fc migrated as a single species having a
molecular size of 91 kD, indicating that the chimeric proteins
assembled as homodimers. Moreover, the IL-10/Fc fusion proteins
bound both anti-mIL-10 mAb (FIG. 2B) and anti-mIgG heavy chain
polyclonal antibodies (FIG. 2A), confirming the cytokine
specificity of the IL-10 moiety and the isotype specificity of the
Fc.gamma.2a domain.
[0076] Standardization of the Biological Activity of rIL-10 and
IL-10/Fc: Using the same RT-PCR strategy and the 5' NotI sense
oligonucleotide primer described above, mIL-10 cDNA with an XbaI
restriction site added 3' to its native termination codon was
cloned into pRc/CMV. This construct was then transiently expressed
in COS cells by the DEAE dextran method and grown in serum-free
UltraCulture media (BioWhittaker Inc.). At day 5, the culture
supernatant was sterilized and stored at -20.degree. C. to provide
a source of recombinant IL-10 (rIL-10). Using a standard curve
based on commercially supplied rIL-10 (PharMingen), IL-10/Fc and
rIL-10 concentrations were determined by ELISA and then by
bioassay. The unit activity based on ELISA corresponded with that
obtained in a standard IL-10 bioassay, which utilized a murine mast
cell line (MC/9) with rIL-4 (PharMingen) as a co-stimulant (see,
e.g., Thompson-Snipes et al., 1991, J. Exp. Med. 173:507).
[0077] In Vitro Characterization of IL-10/Fc: IL-10/Fc functional
activity was assessed in two independent assays. First, the ability
of IL-10/Fc to inhibit IL-6 secretion by LPS-stimulated macrophages
was measured. In this assay, IL-6 levels in supernatants of
cultures of murine monocyte/macrophage PU5-1.8 cells that were
stimulated in the absence or presence of varying doses of rIL-10 or
IL-10/Fc was measured by ELISA (see, e.g., Fiorentino et al., 1991,
J. Immunol. 147:3815). As is shown in FIG. 3, IL-10/Fc inhibits
LPS-induced IL-6 secretion by PU5-1.8 cells in a dose-dependent
manner.
[0078] The ability of IL-10 to enhance IL-4-dependent growth of
MC/9 mast cells was also assayed. In this assay, I measured
incorporation of [.sup.3H]-thymidine into MC/9 cells that were
grown in 100 U/ml of rIL-10 or IL-10/Fc and in the presence or
absence of a neutralizing anti-murine IL-10 mAb (Biosource
International, Camarillo, Calif.; see, e.g., Thompson-Snipes, 1991,
J. Exp. Med. 173:507). Fc.gamma.R I binding assays were performed
with CHO-K1 cells that had been transfected with human Fc.gamma.R I
cDNA. The murine CHO cells which lacked FC.gamma.R I, FC.gamma.R
II, and IL-10 receptors were transfected by electroporation with 20
.mu.g of PvuI- linearized pRc/CMV carrying cDNA encoding human
Fc.gamma.R I. CHO/Fc.gamma.R I cells (5.times.10.sup.5) were washed
twice with FCM buffer (PBS containing 0.1% FCS (BioWhittaker Inc.)
and 0.1% sodium azide) and then incubation with 10 ig/ml of murine
IgG2a (Cappel, West Chester, Pa.), IL-10/Fc, or IL-10/Fc++. After
incubation for 60 minutes on ice, the cells were harvested and
washed in FCM buffer and then incubated with fluorescein conjugated
polyclonal goat-anti-mouse IgG Fc antibody for 60 minutes in the
dark. The cells were washed and stored in a 1% formalin/PBS
solution at 4EC and then analyzed on a FACStar cell sorter
(Becton-Dickinson, San Jose, Calif.). The data presented in FIG. 4
demonstrate that, as was previously noted with rIL-10, IL-10/Fc
enhances the IL-4-dependent growth of the murine mast cell line
MC/9, and this co-stimulatory effect is blocked by a neutralizing
anti-IL-10 mAb. Thus, on a mole for mole basis in terms of IL-10,
IL-10/Fc possesses a biological function equivalent to that of
rIL-10 in these two bioassays.
[0079] Determination of IL-10/Fc Circulating Half-life: To measure
the circulating half-life of IL-10/Fc, the serum concentration of
IL-10/Fc was determined over time following a single bolus
intravenous injection of the chimeric protein into each of six 8-
to 10-week old BALB/c mice (Jackson Laboratory). Serial 100 .mu.l
blood samples were obtained by retro-orbital bleeding at 0.1, 6,
24, 48, 72, and 96 hours after administration of IL-10/Fc to the
mice. Measurements of the IL-10/Fc circulating half-life were made
in an ELISA with a rat-anti-mouse IL-10 mAb as the capture antibody
and a horseradish peroxidase conjugated rat-anti-mouse Fc.gamma.2a
monoclonal antibody as the detection antibody (PharMingen), thus
assuring that this assay was specific for IL-10/Fc and not IL-10 or
mIgG2a. The circulating half-life of IL-10/Fc was determined to be
31 hours (FIG. 5). Thus IL-10/Fc possesses the biological functions
of IL-10 and a prolonged circulating half-life. Furthermore, due to
the specific mutations introduced into the Fc.gamma.2a CH2 domain,
the Fc.gamma.R I binding abilities have been drastically attenuated
(FIG. 6) (see, e.g., Duncan et al., 1988, Nature 332-563). In
addition, I have found that the mutation in the C1q binding site
greatly diminishes the ability of the Fc.gamma.2a domain to
activate complement. Therefore, the ability of IL-10/Fc to support
CDC has been eliminated.
[0080] LPS-induced Septic Shock: To measure the ability of IL-10/Fc
to treat or inhibit septic shock, eight-to ten-week old BALB/c
female mice were treated with IL-10/Fc, rIL-10, mIgG2a, or PBS
prior to intravenous injection of 500 .mu.g LPS into each mouse. A
total of twenty-four animals received either 2,000 U of IL-10/Fc
(12 animals) or 2,000 U of rIL-10 (12 animals) by intraperitoneal
injection 30 minutes before administration of LPS. In a second
experiment, each of 12 animals received 4,000 U of IL-10/Fc or
rIL-10 24 hours before administration of LPS. In control
experiments, animals were treated with equivalent mass
concentrations of mIgG2a (n=6) or equivalent volumes of PBS (n=12)
given 30 minutes before administration of LPS. Survival was the
endpoint measurement.
[0081] As is shown in FIG. 7, a single dose of 500 .mu.g LPS was
uniformly lethal within 72 hours in animals that were treated with
PBS or mIgG2a. Mice that were treated with 2,000 U of rIL-10 or
IL-10/Fc 30 minutes before LPS challenge had a 50% survival rate.
All of the mice that were treated with 4,000 U of rIL-10 24 hours
prior to LPS challenge died, while 50% of mice pretreated with
4,000 U of IL-10/Fc survived. These data indicate that IL-10/Fc and
rIL-10 provide similar levels of protection from the lethal effect
of LPS when they are given 30 minutes prior to an LPS injection. In
contrast to rIL-10, IL-10/Fc confers prolonged protection, even
when it is administered 24 hours prior to challenge with LPS. This
finding is consistent with the longer circulating half-life of
IL-10/Fc relative to rIL-10. Thus, these data indicate that a
chimeric molecule of the invention provides long-term protection
against septic shock in a known animal model of the disease.
[0082] IL-10/Fc Down-regulates Granuloma Formation in
Schistosome-infected Mice: I have also found that IL-10/Fc inhibits
schistosome-induced granuloma formation in animals. In these
studies, female C57BL/6 mice were infected with 60 cercariae of
Schistosoma mansoni (Puetro Rico strain). After four weeks of
infection, the mice were randomly divided into three experimental
groups:
[0083] (i) 5 mice which received no treatment;
[0084] (ii) 5 mice which were treated with mIgG3; and
[0085] (iii) 5 mice which were treated with IL-10/Fc.
[0086] After four weeks of Schistosome infection, mice in groups
(ii) and (iii) were treated by intraperitoneal injection of mIgG3
or IL-10/Fc as follows: 2 .mu.g/mouse on day 1 of treatment, and
then 1 .mu.g/mouse every day for three weeks. After 7 weeks of
Schistosome infection, the mice in all three experimental groups
were sacrificed. The mice livers were then fixed in formalin,
processed with conventional histopathologic techniques, and then
5-im sections were stained with hematoxylin and eosin.
Granulomatous inflammation was assessed quantitatively with
computer-assisted morphometric analysis. As is indicated by FIG. 8,
treatment with IL-10/Fc inhibited granuloma formation in the livers
of mice. Accordingly, the data obtained with this animal model of
Schistosome infection indicate that IL-10/Fc can be used to inhibit
granuloma formation in animals.
[0087] Prevention or Inhibition of Diabetes with IL-10/Fc: The
chimeric IL-10 proteins of the invention are useful for treating or
inhibiting the development of Type I diabetes in a patient. The
following detailed examples employ a well-known animal model of
human diabetes, the non-obese diabetic (NOD) mouse. To study the
effects of IL-10/Fc on diabetes, a total of 30 mice were divided
into three experimental groups:
[0088] (i) 10 mice which received no treatment;
[0089] (ii) 10 mice which were treated with mIgG3; and
[0090] (iii) 10 mice which were treated with IL-10/Fc.
[0091] Treatment was initiated when the mice reached 5 weeks of
age. Treatment of mice in groups (ii) and (iii) involved
intraperitoneal injection of 2 .mu.g of mIgG3 or IL-10/Fc on day 1,
followed by intraperitoneal injection of 1 .mu.g of mIgG3 or
IL-10/Fc every second day until the mice reached 19 weeks of age.
Diabetes was diagnosed with conventional methods and criteria.
[0092] As is indicated in FIG. 9, at least 50% of the control mice
(mIgG3-treated and untreated mice) developed diabetes during the 20
weeks of the study. In contrast, the onset of diabetes during the
course of the study was completely prevented in mice that had been
treated with IL-10/Fc. These data, obtained with an art-recognized
model of human diabetes, indicate that IL-10/Fc is useful for
preventing or inhibiting the onset of diabetes in animals (e.g.,
humans).
[0093] The usefulness of IL-10/Fc in inhibiting the onset of
diabetes was re-affirmed in an additional experiment with NOD mice.
A total of 19 mice were divided into three experimental groups:
[0094] (i) 6 mice which received no treatment;
[0095] (ii) 7 mice which were treated with mIgG2a; and
[0096] (iii) 6 mice which were treated with IL-10/Fc.
[0097] For mice in groups (ii) and (iii), treatment was initiated
when the mice were 6 weeks of age. The treatment regimen included
intraperitoneal injection of 2 .mu.g of mIgG2a or IL-10/Fc on day
1, followed by intraperitoneal injection of 1 .mu.g of mIgG2a or
IL-10/Fc every second day until the mice reached 25 weeks of age.
Diabetes was diagnosed using conventional methods and criteria.
[0098] As is shown in FIG. 10, at least 50% of the mice in the
control groups (untreated mice and mIgG2a-treated mice) developed
diabetes before reaching 25 weeks of age. In marked contrast, none
of the IL-10/Fc-treated mice developed diabetes. At 52 weeks of
age, 84% of the animals which had been treated with IL-10/Fc
continued to have normal glucose levels (i.e., they did not develop
diabetes) despite the cessation of therapy. These data, obtained
with an art-recognized animal model of human diabetes, indicate
that IL-10/Fc is useful for preventing or inhibiting the onset of
diabetes. These data also indicate that IL-10/Fc provides long-term
protection against diabetes.
[0099] Further evidence that IL-10/Fc is useful for treating
diabetes comes from my finding that mice treated with IL-10/Fc bear
suppressor (anti-diabetogenic) lymphocytes. This beneficial effect
has not been reported for mammals treated with the non-chimeric
IL-10 molecule. In this experiment, 25 ten- to twelve-week old male
NOD mice (i.e., the recipients) were irradiated (700 Rads) to
impair their immune systems. The mice were then divided into four
experimental groups:
[0100] (i) 9 recipients which were treated with a mixture of
30.times.10.sup.6 splenic cells from acutely diabetic female NOD
mice and 30.times.10.sup.6 splenic cells from 11-week old untreated
co-donors;
[0101] (ii) 7 recipients which were treated with a mixture of
30.times.10.sup.6 splenic cells from acutely diabetic female NOD
mice and 30.times.10.sup.6 splenic cells from 11-week old co-donors
that had been treated with mIgG3;
[0102] (iii) 5 recipients which were treated with a mixture of
30.times.10.sup.6 splenic cells from acutely diabetic female NOD
mice and 30.times.10.sup.6 splenic cells from 11-week old co-donors
that had been treated with IL-10/Fc; and
[0103] (iv) 4 recipients which were treated with a mixture of
30.times.10.sup.6 splenic cells from acutely diabetic female NOD
mice and 30.times.10.sup.6 splenic cells from 54-week old co-donors
that had been treated with IL-10/Fc.
[0104] Treatment of NOD mice was initiated when the mice reached 5
weeks of age and included intraperitoneal injection of 2 .mu.g of
mIgG3 or IL-10/Fc on day 1 of treatment, followed by injection of 1
.mu.g of mIgG3 or IL-10/Fc every second day until the mice reached
25 weeks of age. Some of the treated mice were used as cell donors
to test for the presence of suppressive type immune phenomena that
could be detected in cell transfer work.
[0105] Diabetes was diagnosed with conventional methods and
criteria. As is indicated in FIG. 11, greater than 85% of NOD mice
that received splenic cells from untreated or mIgG3-treated
co-donors developed diabetes within 25 days of transfer of the
splenic cells. The development of diabetes was inhibited in mice
that had been treated with IL-10/Fc from 11- or 54-week old
co-donors. These data indicate that the onset of diabetes in an
animal can be inhibited by transferring into the animal splenic
cells that have been treated with IL-10/Fc. Thus, not only are the
remissions caused by chimeric IL-10 (e.g., IL-10/Fc) long-lasting
in contrast to the temporary effect seen with non-chimeric IL-10,
the remissions are associated with the formation of
anti-diabetogenic suppressive immune phenomena that have not been
noted with non-chimeric IL-10 treatment.
Treatment or Prevention of Granuloma Formation with IL-4/Fc
[0106] I have found that treatment of mice with IL-4/Fc inhibits
the formation of granulomas that normally are formed when a mammal
is infected with Schistosoma. Accordingly, IL-4/Fc can be used to
inhibit granulomatous inflammation in a patient and to inhibit or
prevent schistosomiasis.
[0107] Genetic Construction of IL-4/Fc: Complementary DNAs for
murine IL-4 and murine Fc.gamma.2a were generated from mRNA
extracted from Con A-stimulated murine splenic cells (C57BL/6J;
Jackson Laboratory, Bar Harbor, Me.) and an IgG2a-secreting
hybridoma (American Type Culture Collection HB129, Rockville, Md.),
respectively, using standard techniques with reverse transcriptase
MMLV-RT (Gibco BRL, Grand Island, N.Y.) and a synthetic
oligo-dT.sub.(12-18) oligonucleotide (Gibco BRL).
[0108] To make the construct for the non-lytic IL-4/Fc
oligonucleotide, site-directed mutagenesis was used to replace Glu
318, Lys 320, and Lys 322 of the C1q binding motif of Fc with Ala
residues. Similarly, Leu 235 was replaced with Glu to inactivate
the Fc.gamma.R I binding site (FIG. 12).
[0109] Expression and Purification of IL-4/Fc: Proper genetic
construction of IL-4/Fc was confirmed by DNA sequence analysis
following cloning of the fusion genes into the eukaryotic
expression plasmid pRc/CMV (Invitrogen, San Diego, Calif.). This
plasmid carries a CMV promoter/enhancer, a bovine growth hormone
polyadenylation signal, and a neomycin resistance gene for
selection against G418. Plasmids carrying the IL-4/Fc fusion genes
were transfected into Chinese Hamster Ovary cells (CHO-K1) by
electroporation (1.5 kV/3 .mu.F/0.4 cm/PBS) and selected in
serum-free Ultra-CHO media (BioWhittaker Inc., Walkerville, Md.)
containing 1.5 mg/ml of G418 (Geneticin, Gibco BRL). After
subcloning, clones which produced the chimeric protein at high
levels were selected by screening supernatants by ELISA for IL-4.
The IL-4/Fc chimeric protein was purified from culture supernatants
by protein-A sepharose affinity chromatography followed by dialysis
against PBS and 0.22 .mu.m filter sterilization. The purified
protein was stored at -20.degree. C. until it was used.
[0110] CTLL-2 Proliferation Assay with IL-4/Fc: The function of the
IL-4 portion of IL-4/Fc was assayed in a conventional CTLL-2
proliferation assay. In such an assay, the addition of IL-4 to a
culture induces the cells to proliferate, and proliferation can be
detected by assaying for the incorporation of [.sup.3H] into the
cells. The data obtained in this optional assay of IL-4/Fc function
indicate that cell proliferation in response to IL-4/Fc
substantially parallels cell proliferation in response to
recombinant IL-4 over a range of concentrations (FIG. 13).
[0111] Determination of IL-4/Fc Circulating Half-life: I have found
that the circulating half-life of IL-4 can be extended by bonding
IL-4 to a non-lytic Fc region of IgG. In this experiment, the
time-related serum concentration of non-lytic IL-4/Fc was
determined following a single bolus intravenous injection (8 mg) of
IL-4/Fc into mice (FIG. 14). Blood samples were obtained by
retro-orbital bleeding of the mice. Non-lytic IL-4/Fc levels were
then measured in an ELISA with rat-anti-mouse IL-4 monoclonal
antibodies as the capture antibodies and horseradish
peroxide-conjugated rat anti-mouse IgG heavy chain monoclonal
antibodies as the detection antibodies. These data indicate that
the circulating half-life of IL-4/Fc is approximately 25 hours.
[0112] IL-4/Fc Inhibits Schistosome Granuloma Formation in vivo:
Female C57BL/6 mice were infected with 60 cercariae of Schistosoma
mansoni (Puetro Rico strain). After four weeks of infection, the
mice were randomly divided into three groups:
[0113] (i) 5 mice which received no treatment;
[0114] (ii) 5 mice which were treated with mIgG3; and
[0115] (iii) 5 mice which were treated with IL-4/Fc.
[0116] After four weeks of Schistosome infection, mice in groups
(ii) and (iii) were treated by intraperitoneal injection of mIgG3
or IL-4/Fc, respectively, as follows: 2,500 U/mouse on day 1 of
treatment, and then 1,250 U/mouse every day for three weeks. After
7 weeks of Schistosome infection, the mice in all three
experimental groups were sacrificed. The mice livers were then
fixed in formalin, processed with conventional histopathologic
techniques, and then 5-.mu.m sections were stained with hematoxylin
and eosin. Granulomatous inflammation was assessed quantitatively
by computer-assisted morphometric analysis. As is indicated by FIG.
15, treatment with IL-4/Fc inhibited granuloma formation in the
livers of mice. Accordingly, the data obtained with this animal
model of Schistosome infection indicate that IL-4/Fc can be used to
inhibit granuloma formation in animals (e.g., humans).
[0117] Other embodiments are within the following claims. For
example, virtually any mutation can be used to disable the
complement-fixing capability of the Fc region of an antibody.
* * * * *